Sorbitol dehydrogenase gene, a novel recombinant DNA, and a process for producing sorbitol dehydrogenase

ABSTRACT

A sorbitol dehydrogenase gene encoding (a) a protein having an amino acid sequence shown in SEQ ID NO: 1; or (b) a protein with sorbitol dehydrogenase activity consisting of an amino acid sequence comprising one or more deletions, substitutions or additions in the amino acid sequence of (a). Further, the invention provides a novel recombinant DNA having the sorbitol dehydrogenase gene inserted into a vector DNA, and a transformant or a transductant containing the recombinant DNA. Also, the invention provides a sorbitol dehydrogenase production method comprising the steps of: culturing the transformant or the transductant in a medium; and collecting sorbitol dehydrogenase from the culture. According to the present invention, sorbitol dehydrogenase can be produced efficiently.

BACKGROUND OF THE INVENTION

[0001] 1. Field of the Invention

[0002] This invention relates to a gene of sorbitol dehydrogenase whichproduces D-fructose by oxidizing D-sorbitol in the presence of NAD+ andwhich produces, through a reverse reaction of the above reaction,D-sorbitol by reducing D-fructose in the presence of NADH, a novelrecombinant DNA, and a method for producing sorbitol dehydrogenase.

[0003] 2. Description of the Related Art

[0004] D-sorbitol exists in trace amounts in blood serum, urine or thelike of humans, and it is known that its content is an importantindicator in the diagnosis of diseases, for example, diabetes. Now atresearch institutes and so on, an enzyme method is used for thedetermination of D-sorbitol in blood serum.

[0005] Further, by hydrogenating glucose under high temperature andpressure, D-sorbitol solution is obtained, and then D-sorbitol powder orgranule is obtained by refining the obtained D-sorbitol solution by useof ion-exchange resin method or the like. D-sorbitol is odorless but ithas a brisk sweetness, and thereby brings coolness to the tongue or ithas an excellent hygroscopicity. Due to these properties, it is widelyused as a sweetener for chewing gum, candy, and other confectionary, asa robustness-imparting material for synthetic alcohol, or as a sweetenerand humectant for tobacco. In addition, D-fructose can be obtained bymethods such as a method of hydrolyzing a plant like Heliantustuberosus, which contains inulin in abundance, and a method ofconverting sucrose by the action of invertase. D-fructose has a delicatebut strong sweetness, and imparts wettability. Thus, it is widely usedas a sweetener or the like in the food industry.

[0006] In view of the foregoing, it is of industrially importantsignificance to obtain an enzyme usable for determining D-sorbitolexisting in trace amounts in blood serum, urine or the like, andD-sorbitol or D-fructose contained in foods.

[0007] Sorbitol dehydrogenase has conventionally been produced, forinstance by inoculating Pseudomonas sp. in a medium, culturing it, andcollecting the culture product. (refer to Japanese Patent No. 3152855)

[0008] However, the above production method of sorbitol dehydrogenasehas some drawbacks such as insufficient yield.

SUMMARY OF THE INVENTION

[0009] In order to solve these problems of the prior art, it is anobject of the present invention to provide a sorbitol dehydrogenasegene, a novel recombinant DNA thereof, and a method for producingsorbitol dehydrogenase.

[0010] As a result of various reviews on the above problems, theinventors of the present invention have successfully accomplishedisolation of a sorbitol dehydrogenase gene derived from Pseudomonas sp.KS-E1806, and determination of the structure thereof. Further, theinventors have obtained a recombinant DNA having a gene encodingsorbitol dehydrogenase inserted into a vector DNA, and it has been foundthat after this recombinant DNA is incorporated into a strain belongingto the genus Escherichia, and the recombinant-DNA-containing strain withsorbitol dehydrogenase production ability is cultured, sorbitoldehydrogenase can effectively be produced. Based on these findings, theinventors accomplished the present invention.

[0011] In accordance with a first aspect of the present invention, thereis provided a sorbitol dehydrogenase gene encoding the following protein(a) or (b): (a) a protein having an amino acid sequence shown in SEQ IDNO: 1; or (b) a protein which consists of an amino acid sequence havingone or more amino acid deletions, substitutions or additions relative tothe amino acid sequence of (a), and has sorbitol dehydrogenase activity.

[0012] In accordance with a second aspect of the present invention,there is provided a sorbitol dehydrogenase gene comprising the followingDNA (a) or (b): (a) a DNA having a nucleotide sequence shown in SEQ IDNO: 2; and (b) a DNA encoding a protein which hybridizes with the DNAhaving a nucleotide sequence complementary to the DNA having anucleotide sequence of (a) under stringent conditions, and has sorbitoldehydrogenase activity.

[0013] In accordance with a third aspect of the present invention, thereis provided a novel recombinant DNA wherein the above sorbitoldehydrogenase gene has been inserted into a vector DNA.

[0014] In accordance with a fourth aspect of the present invention,there is provided a transformant or transductant including the abovenovel recombinant DNA.

[0015] In accordance with a fifth aspect of the present invention, thereis provided a method for producing sorbitol dehydrogenase, comprisingthe steps of:

[0016] culturing the transformant or the transductant in a medium; and

[0017] collecting sorbitol dehydrogenase from the medium.

DETAILED DESCRIPTION OF THE INVENTION

[0018] The present invention will hereinafter be described in detail.

[0019] For example, a sorbitol dehydrogenase gene of the presentinvention can be isolated as follows.

[0020] First, Pseudomonas sp. KS-E1806 is cultivated, e.g. by a methoddescribed in Japanese Patent No. 3152855, and then chromosomal DNA isextracted from the obtained microorganism strain. The chromosomal DNAcan be prepared from cell bodies of the strain, e.g. by a methoddescribed in Current Protocols in Molecular Biology (WILEY Interscience,1989).

[0021] Next, the above microorganism is cultivated e.g. by a methoddescribed in Japanese Patent No. 3152855, and sorbitol dehydrogenase ispurified, thereby obtaining sorbitol dehydrogenase.

[0022] The obtained sorbitol dehydrogenase is subjected tolysylendopeptidase (available from Wako Pure chemical Industries, Ltd.)treatment under a degeneration condition for fragmentation. The abovefragment is fractionated, for example, by reverse phase high performanceliquid chromatography using Capsulepack C18UG300 column (Shiseido Co.,Ltd.), and amino acid sequences with regard to several kinds of fragmentpeptides are determined, e.g. by Procise 492 protein sequencer(manufactured by Applied Biosystems Japan Ltd.).

[0023] Based on the above amino acid sequences, a primer for polymerasechain reaction (hereinafter abbreviated as PCR) is prepared. At thistime, considering codon degeneracy, while a nucleotide mixture is usedat a position where there is a codon degeneracy of not greater than twokinds of nucleotides, inosine is used at a position where there is acodon degeneracy of 3 to 4 kinds of nucleotides. Then, a long primerwith a length of not less than 40 mer, preferably 42 to 44 mer, isprepared. PCR is conducted with the prepared primer using chromosomalDNA of the previously obtained Pseudomonas sp. KS-E1806 as a template.The amplified DNA fragment is incorporated into a vector DNA, such aspCR2.1 vector (available from Invitorogen Japan K.K.), affording arecombinant plasmid. A nucleotide sequence of the inserted DNA in theplasmid is determined, e.g. by a Multi Capirally DNA ayalysis systemCEQ2000 (manufactured by Beckman Coulter, Inc.), then a DNA having atboth ends a nucleotide sequence correctly encoding the amino acidsequence of the peptide used for designing PCR primer is selected. Thethus obtained amplified DNA fragment is a part of the sorbitoldehydrogenase gene of the present invention (partial gene).

[0024] The above partial gene is labeled, and a clone containing atarget gene is isolated by hybridization from a library containing thechromosomal fragment of Pseudomonas sp. KS-E1806. DNA fragment labelingand hybridization detection can be carried out, e.g. by DIG system(manufactured by Roche Diagnostics K.K.).

[0025] The library can be constructed, for example, as follows. First,the chromosomal DNA is completely digested by a restriction enzyme suchas EcoRI, and then subjected to conventional agarose gel electrophoresisand Southern blot analysis using the above PCR product as a probe. As aresult, the chain length of the DNA fragment containing sorbitoldehydrogenase gene is determined.

[0026] Next, chromosomal DNA is completely digested by a restrictionenzyme above such as EcoRI, and subjected to agarose gelelectrophoresis, thereafter a gel containing the determined chain lengthof DNA fragment is excised. Then, DNA in the excised gel is collected byGENE CLEAN II (manufactured by Funakoshi Co., Ltd.), and incorporatedinto a vector DNA in accordance with a conventional method. Examples ofthe vector DNA to be used include a plasmid DNA such as pUC119(available from Takara Shuzo Co., Ltd.) and pBR322 (available fromTakara Shuzo Co., Ltd.), and a bacteriophage DNA such as λENBL3(available from Stratagene) and λDASH II (available from Funakoshi Co.,Ltd.).

[0027] By use of the recombinant DNA thus obtained, for example,Escherichia coli K-12, preferably Escherichia coli JM109 (available fromTakara Shuzo Co., Ltd.), DH5α (available from Takara Shuzo Co., Ltd.) istransformed, or the recombinant DNA is transduced thereinto, so as toobtain a transformant or a transductant. Examples of a host cell to beused, in addition to a cell of Escherichia coli, include yeast, afungus, actinomyces, and an animal cell.

[0028] This transformation can be carried out, e.g. by a method of D. M.Morrison (Method in Enzymology, 68, 326-331, 1979). Further, thetransduction can be carried out, e.g. by a method of B. Hohn (Method inEnzymology, 68, 299-309, 1979).

[0029] In order to obtain a purified recombinant plasmid DNA from theisolated transformant (containing sorbitol dehydrogenase gene therein),QIAGEN Plasmid Midi Kit (available from Qiagen K.K.), for example, canbe used.

[0030] By using the purified recombinant plasmid DNA, the wholenucleotide sequence of sorbitol dehydrogenase gene is analyzed by meansof a method explained in process (5) of the Examples describedhereinafter, then determining amino acid sequence of the polypeptidetranslated from the gene having the above nucleotide sequence therein.

[0031] This amino acid sequence is as shown in SEQ ID NO: 1. Thesorbitol dehydrogenase gene of the present invention is a gene encodingthe amino acid sequence thus determined.

[0032] Incidentally, as far as a sorbitol dehydrogenase gene encodes anamino acid sequence comprising one or more, preferably several aminoacid deletions, substitutions, or additions relative to the amino acidsequence shown in SEQ ID NO: 1, and having sorbitol dehydrogenaseactivity, such a sorbitol dehydrogenase gene is included in the presentinvention.

[0033] Moreover, as far as there is obtained a sorbitol dehydrogenasegene encoding an amino acid sequence comprising one or more amino aciddeletions, substitutions, or additions relative to the amino acidsequence shown in SEQ ID NO: 1, and having a sorbitol dehydrogenaseactivity, any method may be applied therefor. Examples thereof includesite-specific mutation induction which is a well-known method to cause apoint mutation or a deletion mutation on a gene; a method comprising ofselectively cleaving a gene, deleting or adding a selected nucleotide,and ligating the gene; and an oligonucleotide mutation induction method.

[0034] These DNAs are highly likely to encode a polypeptide havingsorbitol dehydrogenase activity, and it is possible to producetransformants and select transformants having activity.

[0035] In order to obtain a gene substantially identical to the sorbitoldehydrogenase gene of the present invention, DNA encoding a polypeptidehaving sorbitol dehydrogenase activity can be selected by performinghybridization under stringent conditions with a DNA having thenucleotide sequence of SEQ ID NO: 2, a chain complementary thereto, or aprobe containing a fragment thereof. Herein, stringent conditions meanconditions wherein only a specific hybrid is selectively formed,enabling signal detection, yet a non-specific hybrid is not produced.Although such conditions are slightly different depending on individualbiological species, conditions can easily be determined by examiningseveral salt concentrations or temperatures for hybridization, andwashing by means of a conventional method. As these conditions, forexample, since a specific signal is observed in items (3) and (4) of theExamples described hereinafter, the hybridization is conducted at atemperature of 37 to 42° C. overnight by use of DIG Easy Hyb reagent(available from Roche Diagnostics K.K.). The washing is carried outtwice with 0.5×SSC, 0.1% SDS for 15 minutes. The washing is carried outat a temperature of not less than 45° C., preferably not less than 52°C., more preferably not less than 57° C. Although a DNA which can behybridized under such conditions is highly likely to encode a peptidehaving sorbitol dehydrogenase activity, DNA containing a variationcausing a loss of sorbitol dehydrogenase activity may be included.However, such variation-containing DNA can be removed, aftertransformation, by determining the sorbitol dehydrogenase productionability of the transformant.

[0036] The recombinant DNA thus obtained containing a sorbitoldehydrogenase gene does not have a promoter for attaining a strongexpression in E. coli, and thus the transformant strain having therecombinant DNA has a low productivity of sorbitol dehydrogenase. Then,a strain with a high productivity of sorbitol dehydrogenase is obtainedby the following operation.

[0037] First, based on the nucleotide sequence obtained in the aboveoperation, oligonucleotides comprising N-terminus and the C-terminus ofsorbitol dehydrogenase gene, respectively, and about 12 nucleotidesbefore and after N- or C-terminus (the oligonucletide comprising intotal 30 nucleotides; the oligonucleotide at C-terminus is acomplementary strand) are synthesized, and the synthesizedoligonucleotides are used as primers. NdeI sites have been incorporatedinto the synthesized primers such that a coding region is obtained afteramplified product by PCR is digested by application of NdeI (availablefrom Takara Shuzo Co., Ltd.). In other words, coding region of sorbitoldehydrogenase can be obtained by carrying out PCR using the purifiedrecombinant plasmid DNA obtained above as a template followed bydigesting the resulting product by NdeI.

[0038] The obtained DNA is inserted into a vector DNA having a DNAsequence containing an expression region such as a promoter, anoperator, and a ribosome binding site derived from E. coli lactoseoperon (See The Operon, p. 227, Cold Spring Harbor Laboratory, 1980).The vector DNA to be used may be a plasmid DNA or a bacteriophage DNA.For example, a vector pUTE500K′ (disclosed in Japanese PatentApplication Laying-Open (kokai) No. 08-205861) described in process (6)of Examples can be used. Using the obtained recombinant DNA, forexample, E. coli K-12, preferably E. coli JM109 (available from TakaraShuzo Co., Ltd.), or DH5α (available from Takara Shuzo Co., Ltd.), istransformed, or the recombinant DNA is transduced thereinto, therebyeach affording a strain.

[0039] The production of sorbitol dehydrogenase by use of the aboveobtained transformant or transductant, for example, a strain belongingto the genus Escherichia, each having the ability for producing asorbitol dehydrogenase, can be carried out as follows. For culturing theabove microorganism, a conventional solid culture method may be used.However, it is preferable to use a liquid culture method for culturingas much as possible.

[0040] Additionally, as a medium for culturing the above microorganism,there may be used a medium prepared by adding at least one mineral salt,such as potassium dihydrogenphosphate, dipotassium hydrogenphosphate,magnesium sulfate, ferric chloride, ferric sulfate, and manganesesulfate; further if necessary, carbohydrate, vitamin, or the like; to atleast one nitrogen source, such as a yeast extract, peptone, meatextract, corn steep liquor, or an exudation of soybean or wheat malt.

[0041] Further, it is appropriate to adjust initial pH of the medium to7 to 9. Further, the culture is conducted for 6 to 24 hours at atemperature of from 30 to 42° C., preferably about 37° C. by submergedculture with aeration-agitation, shake-culture, stationary culture, orthe like. After the culture is finished, a conventional enzymecollection means may be used for collecting sorbitol dehydrogenase fromthe culture product.

[0042] Bacterial bodies are separated from the culture product, forexample, by filtration, centrifugation or the like, and washed. It ispreferred to collect sorbitol dehydrogenase from the bacterial bodies.In this case, the bacterial bodies per se may be used. However, it ispreferable to collect sorbitol dehydrogenase from the bacterial bodiesby a method of disrupting the bacterial bodies by using a disruptingmeans such as a ultrasonic disruptor, French press, and a dyna-mill, amethod of digesting cell wall of the bacterial bodies by using cell walllysing enzyme such as lysozyme, and a method of extracting an enzymefrom the bacterial bodies by using a surfactant such as Triton X-100.

[0043] For isolating sorbitol dehydrogenase from the crude enzymesolution thus obtained, a conventional method used for enzymepurification may be used. Examples of conventional methods includeammonium sulfate salting-out, organic solvent precipitation,ion-exchange chromatography, gel filtration chromatography, adsorptionchromatography, electrophoresis, and these may preferably be used incombination as required.

[0044] The obtained sorbitol dehydrogenase has physico-chemicalproperties as shown below.

[0045] (1) Function: to oxidize, in the presence of NAD+, D-sorbitol toproduce D-fructose and NADH, and by the reverse reaction thereof, toreduce, in the presence of NADH, D-fructose to produce D-sorbitol andNAD+.

[0046] (2) Substrate specificity: to react specifically to D-sorbitoland galactitol.

[0047] (3) Optimum pH and stable range of pH: an optimum pH is about 10,and a stable pH range is from 5.5 to 10.5 in the case of 4-hourtreatment at 30° C.

[0048] (4) Optimum reaction temperature range: in the case of 100 mMTris-HCl buffer solution (pH 9.0), the optimum reaction temperaturerange is about 50° C.

[0049] (5) Deactivation condition by pH, temperature, etc.: in the caseof 4-hour treatment at 30° C., sorbitol dehydrogenase is kept stable atpH of 5.5 to 10.5, and at a pH of not greater than 5.0 and not less than11.0 the sorbitol dehydrogenase is completely deactivated. In the caseof 30-minute treatment at pH of 7.0, sorbitol dehydrogenase is keptstable at a temperature of up to around 40° C. In case of 40° C.treatment at pH of 7.0, 24 hours are enough for complete deactivation.

[0050] (6) Inhibition: HgCl₂ has a strong inhibitory action.

[0051] (7) Molecular weight: about 64,500±5,000 (gel filtration)

[0052] (8) Assay of titer: the titer of the enzyme is assayed by thefollowing method, and an amount of the enzyme for producing 1 μmol ofNADH for one minute is taken as 1U.

[0053] (Preparation of Reagents)

[0054] First solution; substrate solution, 20 g of D-sorbitol isdissolved in distilled water, and filled to 100 ml.

[0055] Second solution; substrate solution, 86 mg of NAD+ is dissolvedin distilled water, and filled to 4 ml.

[0056] Third solution; buffer solution, 6.05 g oftris(hydroxymethyl)aminomethane is dissolved in distilled water, and pHis adjusted to 9.0 by 4N HCl, then filled to 500 ml.

[0057] (Determination Procedure)

[0058] 1) 0.5 ml of the first solution, 0.05 ml of the second solution,and 2.4 ml of the third solution are mixed with each other andpre-incubated for 5 minutes at 37° C.

[0059] 2) The 5-minute pre-incubated solution and 0.05 ml of an enzymesolution adjusted to 0.1 to 0.8 U/ml are mixed with each other, and theincreased value of absorbance of a sample per 1 minute is measured at37° C. at a wavelength of 340 nm. 3) For determination of a blank value,0.5 ml of distilled water is added instead of the first solution, and0.05 ml of the second solution and 2.4 ml of the third solution aremixed therewith, and pre-incubated for 5 minutes at 37° C. Then, 0.05 mlof the enzyme solution adjusted to 0.1 to 0.8 U/ml are mixed with thepre-incubated solution, and the increased value of absorbance of theblank per 1 minute is measured at 37° C. at a wavelength of 340 nm.

[0060] (Calculation of titer value)

U/ml=[{(change of absorbance value of sample—increase of absorbancevalue of blank)×3}/(6.22×0.05)]×dilution factor

[0061] The enzyme acts on D-sorbitol, and has stable enzyme activityeven under conditions, which are often applied for measurement accordingto a conventional enzyme method (reaction at pH 9.0, 37° C., for 30minutes). By use of the enzyme, for example, the amount of D-sorbitolpresent in human blood serum or urine, or foods can be determined withexcellent accuracy. Further, by the reverse reaction of the enzyme, forexample, the amount of D-fructose in foods can be determined.

EXAMPLES

[0062] Hereinafter, the present invention is described in more detail byreference to the Examples, which, however, are not intended to limit thescope of the present invention.

[0063] (1) Preparation of a chromosomal DNA of Pseudomonas sp. KS-E1806

[0064] Pseudomonas sp. KS-E1806 (FERM BP-7616) (converted from FERMP-14299) was inoculated onto 100 ml of a enzyme production medium [0.5%of D-sorbitol, 2.0% of polypepton, 0.5% of yeast extract, 0.01% ofKH₂PO₄, 0.01% of K₂HPO₄, 0.01% of MgSO₄·7H₂O, and tap water (at pH of7.2)], and shake-cultured at 30° C. for about 20 hours, then collectingbacterial bodies by centrifugation. From the bacterial bodies 1.2 mg ofchromosomal DNA was obtained by use of G NOME DNA Isolation Kit(available from Funakoshi Co., Ltd.).

[0065] (2) Acquisition of partial gene

[0066] Next, sorbitol dehydrogenase was purified from the abovemicroorganism by a method described in Japanese Patent No. 3152855, and50 μg of the obtained sorbitol dehydrogenase was subjected tolysylendopeptidase (Wako Pure Chemical Industries, Ltd.) treatment underdenaturing conditions, resulting in fragmentation. The resultantfragments were fractionated and collected by reversed phase HPLC usingWaters μBondapack C18 (available from Millipore Corporation), then aminoacid sequences of several kinds of fragmental peptides were determinedby use of Procise 492 protein sequencer (manufactured by AppliedBiosystems Japan Ltd.).

[0067] In accordance with the above internal amino acid sequence, aplurality of PCR primers were prepared, and PCR was conducted by use ofExTaq DNA polymerase (available from Takara Shuzo Co., Ltd.) with thechromosomal DNA prepared in process (1) used as a template. First, 15-26mer primers were plurally prepared by using the nucleotide mixture at aposition where there is a codon degeneracy. Although PCR was conductedwith various combinations of these primers, annealing temperatures beingreviewed, a target partial gene could not be obtained. Therefore, longprimers, in which a sense primer is 42 mer [an internal amino acidsequence of the obtained protein: a polynucletide corresponding to ValAsn Gly Ile Ala Pro Gly Val Val Asp Thr Pro Met Trp:5′-GTIAAYGGIATIGCICCIGGIGTIGTITGYACICCIATGTGG-3′ (A: adenine, C:cytosine, G: guanine, T: thymine, I: inosine, Y: thymine or cytosine)]and an antisense primer is 44 mer mer [an internal amino acid sequenceof the obtained protein: a polynucletide corresponding to Asp Ala AspTyr Ile Thr Ala Gln Thr Leu Asn Val Asp Gly Gly:5′-CCICCRTCIACRTTIAIIGTYTGIGCIGTIATRTARTCIGCRTC-3′ (A: adenine, C:cytosine, G: guanine, T: thymine, I: inosine, R: adenine or guanine, Y:thymine or cytosine)], were then prepared by using the nucleotidemixture at a position where there is a codon degeneracy of one or twokinds of nucleotides, and inosine at a position where there is a codondegeneracy of three to four kinds of nucleotides. By use of the preparedprimers, annealing at 64° C. and 60 cycles of PCR reactions wereconducted, and subsequently a plurality of gene fragments wereamplified.

[0068] Among them, four major DNA fragments were incorporated intopCR2.1 vector (available from Invitrogen Japan K.K.), so that arecombinant plasmid was obtained. A nucleotide sequence of the insertedDNA in the plasmid was determined by a multi capillary DNA analysissystem CEQ2000 (manufactured by Beckman Coulter, Inc.). As a result, thelongest DNA fragment (227 bp) of the four fragments had at both endsthereof nucleotide sequences correctly encoding amino acid sequences ofthe peptides used for designing the PCR primers. In addition, in theamino acid sequence deduced based on the determined nucleotide sequence,there existed the internal amino acid sequence determined by the proteinsequencer. Accordingly, it was proved the amplified DNA fragment thusobtained was a part of the sorbitol dehydrogenase gene (partial gene) ofthe present invention.

[0069] (3) Construction of chromosomal DNA library

[0070] First, 2 μg of the chromosomal DNA were completely digested byEcoRI, and thereafter subjected to a conventional agarose gelelectrophoresis, further to Southern blot analysis with the above 227 bpPCR product as a probe. The probe was labeled with digoxigenin usingDIG-High Prime (available from Roche Diagnostics K.K.). As a result, itwas confirmed that the EcoRI fragment containing a sorbitoldehydrogenase gene had a chain length of about 1.3 kb. Then, 10 μg ofthe chromosomal DNA was completely digested by a restriction enzymeEcoRI, and subjected to agarose gel electrophoresis.

[0071] Next, after excising gel containing the EcoRI fragment with achain length of 1.3 kb, DNA in the gel was collected by GENE CLEAN II(manufactured by Funakoshi Co., Ltd.). The DNA and EcoRI digest of 100ng of a plasmid vector DNA pUC19 (available from Takara Shuzo Co., Ltd.)were ligated with one unit of T4 DNA Ligase (available from RocheDiagnostics K.K.). The obtained recombinant plasmid DNA was used fortransforming Escherichia coli DH5α strain (available from Takara ShuzoCo., Ltd.) in accordance with a method of D. M. Morrison (Methods inEnzymology, 68, p. 326-331, 1979). As a consequence, 2000 colonies wereobtained, and blotted on a nylon membrane filter Hybond-N+ (availablefrom Amersham Pharmacia Biotech K.K.) according to the protocol ofAmersham Pharmacia Biotech K.K.

[0072] (4) Isolation of sorbitol dehydrogenase gene by colonyhybridization

[0073] A sorbitol dehydrogenase gene was obtained by screening thechromosomal DNA library constructed in process (3) with 227 bp PCRproduct obtained in process (2) as a probe. DIG High Prime (availablefrom Roche Diagnostics K.K.) was used to label the probe withdigoxigenin. In the screening, colony hybridization was conducted, inaccordance with a method described in Current Protocols in MolecularBiology (WILEY Interscience, 1988), thereby obtaining four positivecolonies. From the four positive colonies, plasmid DNA was prepared byQIAGEN Plasmid Midi Kit (available from Qiagen K.K.).

[0074] (5) Analysis of sorbitol dehydrogenase gene

[0075] With respect to the EcoRI fragment inserted into the plasmid DNAobtained from the four positive colonies, about 200 bp of nucleotidesequences from both ends thereof were analyzed by Multi Capillary DNAAnalysis System CEQ2000 (manufactured by Beckman Coulter, Inc.), andthereafter all of them were considered the same clone. Results ofdetermining the entire nucleotide sequence of the inserted fragmentsshowed that the full length of a sorbitol dehydrogenase gene wasincluded. The determined nucleotide sequence of the sorbitoldehydrogenase gene, and the amino acid sequence of the polypeptidetranslated from the DNA sequence are shown in SEQ ID NO: 2 and SEQ IDNO: 1, respectively. It was proved that an ORF of the sorbitoldehydrogenase gene comprises 774 bp, 258 amino acids.

[0076] (6) Preparation of Escherichia coli DH5α (pSDH1)

[0077] A DNA consisting of a coding region for soribtol dehydrogenasewas prepared by the following method. Prepared as PCR primers were anoligonucleotide comprising the N-terminus of the sorbitol dehydrogenasegene and about 12 nucleotides each before and after the N-terminus (SEQID NO: 3) and an oligonucleotide comprising the C-terminus thereof andabout 12 nucleotides each before and after the C-terminus (SEQ ID NO:4). These primers were designed to have an NdeI site incorporated in thesequence thereof such that the coding region is obtained by digesting aPCR amplified product with NdeI.

[0078] Using these primers, PCR was conducted with the plasmid DNAobtained from the positive colonies as a template, and therebyamplifying the DNA containing a region encoding sorbitol dehydrogenase.After digesting the DNA with NdeI, the DNA was inserted into the NdeIsite of pUTE500K′ (Japanese Patent Application Laying-Open (kokai) No.08-205861), an expression plasmid vector having a DNA sequencecontaining an expression region such as promoter, operator and aribosome-binding site derived from E. coli lactose operon (refer to TheOperon, p. 227, Cold Spring Harbor Laboratory, 1980), consequentlyaffording a recombinant plasmid pSDH1 DNA.

[0079] In accordance with a method of D. M. Morrison (Methods inEnzymology, 68, p. 326-331, 1979), E. coli DH5α (available from TakaraShuzo Co., Ltd.) was transformed by use of the recombinant plasmid DNApSDH1 thereby to obtain a transformant strain, E. coli DH5α (pSDH1).Incidentally, the E. coli DH5α (pSDH1) was deposited as FERM BP-7615 atthe International Patent Organism Depository of the IndependentAdministrative Institution National Institute of Advanced IndustrialScience and Technology.

[0080] After the obtained E. coli DH5α (pSDH1) was shake-cultured for 16hours in a TY medium (1% of bacto trypton, 0.5% of bacto yeast extract,0.5% of NaCl, pH 7.0) containing 1 mMisopropyl-β-D-thiogalactopyranoside, sorbitol dehydrogenase activity wasmeasured and it was found to be 11.4 U/ml.

EFFECT OF THE INVENTION

[0081] According to the present invention, sorbitol dehydrogenase can beproduced efficiently.

SEQUENCE LISTING

[0082]

1 8 1 258 PRT Pseudomonas sp. KS-E1806 1 Met Arg Leu Glu Asp Lys Val AlaIle Leu Thr Gly Ala Ala Ser Gly 1 5 10 15 Ile Gly Glu Ala Val Ala GlnArg Tyr Leu Asp Glu Gly Ala Arg Cys 20 25 30 Val Leu Val Asp Val Lys ProAla Gly Gly Ser Leu Ala Arg Leu Ile 35 40 45 Glu Ala Asn Pro Gly Arg AlaVal Ala Val Thr Ala Asp Val Thr Arg 50 55 60 Arg Asp Asp Ile Thr Arg IleVal Ala Thr Ala Val Glu Arg Phe Gly 65 70 75 80 Gly Val Asp Ile Leu PheAsn Asn Ala Ala Leu Phe Asp Met Arg Pro 85 90 95 Leu Leu Asp Glu Ser TrpAsp Val Phe Asp Arg Leu Phe Ser Val Asn 100 105 110 Val Lys Gly Leu PhePhe Leu Met Gln Ala Val Ala Gln Arg Met Val 115 120 125 Glu Gln Gly ArgGly Gly Lys Ile Val Asn Met Ser Ser Gln Ala Gly 130 135 140 Arg Arg GlyGlu Ala Leu Val Ser His Tyr Cys Ala Thr Lys Ala Ala 145 150 155 160 ValIle Ser Tyr Thr Gln Ser Ala Ala Leu Ala Leu Ala Pro His Arg 165 170 175Ile Asn Val Asn Gly Ile Ala Pro Gly Val Val Asp Thr Pro Met Trp 180 185190 Glu Gln Val Asp Ala Leu Phe Ala Arg Tyr Glu Asn Arg Pro Leu Gly 195200 205 Glu Lys Lys Arg Leu Val Gly Glu Ala Val Pro Leu Gly Arg Met Gly210 215 220 Val Pro Gly Asp Leu Thr Gly Ala Ala Leu Phe Leu Ala Ser AlaAsp 225 230 235 240 Ala Asp Tyr Ile Thr Ala Gln Thr Leu Asn Val Asp GlyGly Asn Trp 245 250 255 Met Ser 2 774 DNA Pseudomonas sp. KS-E1806 2gtgagactgg aagacaaggt cgcgatcctg acgggcgccg caagcggcat cggcgaggcg 60gtcgcacaac gctatctgga cgagggcgcg cgctgcgtgc tcgtcgacgt gaagccggca 120ggcggctcgc tcgcgcggct gatcgaggcc aacccgggcc gcgcggtggc cgtcacggcc 180gacgtcacgc gtcgcgacga catcacgcgg atcgtcgcca cggcggtcga gcgcttcggc 240ggcgtcgaca ttctgttcaa caacgcggcg ctgttcgaca tgcgtccgct cctcgatgaa 300tcctgggacg tgttcgaccg gctgttctcg gtcaacgtga aagggctgtt cttcctgatg 360caggcggttg cgcaacggat ggtcgagcag gggcgcggcg gcaagatcgt caacatgtcg 420tcgcaggccg gccgtcgcgg cgaggcgctc gtttcgcact actgcgcgac caaggccgcg 480gtgatcagct atacgcagtc ggccgcgctc gcgctcgcgc cgcaccggat caacgtgaac 540ggcatcgcgc cgggcgtggt cgacacgccg atgtgggagc aggtcgatgc gctgttcgcg 600cgctacgaga accggccgct cggcgagaag aagcggctcg tcggtgaagc cgtgccgctc 660ggccgcatgg gcgtgccggg cgacctgacg ggcgccgcgc tgttcctcgc gtcggccgat 720gccgactaca tcaccgccca gacgttgaac gtcgatggcg gcaactggat gagc 774 3 30 DNAArtificial Sequence Synthetic 3 aaccggagac agcatatgag actggaagac 30 4 30DNA Artificial Sequence Synthetic 4 cttgcgagcg gccatatgtc agctcatcca 305 14 PRT Artificial Sequence Synthetic 5 Val Asn Gly Ile Ala Pro Gly ValVal Asp Thr Pro Met Trp 1 5 10 6 42 DNA Artificial Sequence Synthetic 6gtnaayggna tngcnccngg ngtngtntgy acnccnatgt gg 42 7 15 PRT ArtificialSequence Synthetic 7 Asp Ala Asp Tyr Ile Thr Ala Gln Thr Leu Asn Val AspGly Gly 1 5 10 15 8 44 DNA Artificial Sequence Synthetic 8 ccnccrtcnacrttnanngt ytgngcngtn atrtartcng crtc 44

What is claimed is:
 1. A sorbitol dehydrogenase gene encoding thefollowing protein (a) or (b): (a) a protein having an amino acidsequence shown in SEQ ID NO: 1; or (b) a protein which consists of anamino acid sequence comprising one or more amino acid deletions,substitutions or additions relative to the amino acid sequence of (a),and has sorbitol dehydrogenase activity.
 2. A sorbitol dehydrogenasegene comprising the following DNA (a) or (b): (a) a DNA having anucleotide sequence shown in SEQ ID NO: 2; or (b) a DNA encoding aprotein which hybridizes with the DNA having a nucleotide sequencecomplementary to the DNA having a nucleotide sequence of (a) understringent conditions, and has sorbitol dehydrogenase activity
 3. A novelrecombinant DNA, wherein the sorbitol dehydrogenase gene of claim 1 or 2is inserted into a vector DNA.
 4. A transformant or a transductantincluding the novel recombinant DNA of claim
 3. 5. A method forproducing sorbitol dehydrogenase, comprising the steps of: culturing thetransformant or the transductant of claim 4 in a medium; and collectingsorbitol dehydrogenase from the culture.